The human immunodeficiency virus (HIV) is a member of the lentivirus family of animal retroviruses. Lentiviruses, including visna virus of sheep, and the bovine, feline and simian immunodeficiency viruses (SIV), are capable of long-term latent infection in cells and short-term cytopathic effects, and they all produce slowly progressive, fatal diseases, which include wasting syndromes and central nervous system degeneration. Two closely related types of HIV, designated HIV-1 and HIV-2, have been identified. HIV-1 and HIV-2 differ in genomic structure and antigenicity, sharing only 40 percent nucleic acid sequence homology. Nonetheless, both forms of HIV cause similar clinical syndromes.
HIV infection ultimately results in impaired function of both the specific and innate immune systems. The most prominent defects are in cell-mediated immunity and they can be attributed to a lack of CD4+ T cells, and/or abnormalities in immune system function with normal CD4+ T cell counts. The hallmark of the progression of HIV-induced disease is the diminishing number of CD4+ T cells in the peripheral blood, from a normal of about 1000/mm3 to less than 100/mm3 in fully developed AIDS. Since CD4+ helper T cells are essential for both cell-mediated and humoral immune responses to various microbes, the loss of these lymphocytes is a major reason why AIDS patients become susceptible to so many infections. The loss of CD4+ cells may occur after several months or take longer than 10 years in different individuals.
HIV gene expression may be divided into an early stage, during which regulatory genes are expressed, and a late stage, during which structural genes are expressed and full-length viral genomes are packaged. Expression of the early regulatory genes, including Tat, requires sequential RNA splicing events in the nucleus that generate short mRNAs, which are then transported to the cytoplasm and are subsequently translated into proteins. The Tat gene encodes a protein that binds to a sequence present in the long terminal repeats (LTR) called the transactivating response element (TAR), resulting in enhanced viral gene expression. Binding of the Tat protein to the viral LTR causes a 1000-fold increase in RNA polymerase II-catalyzed transcription of the provirus, and is required for replication of the virus (Abbas, A. K. et al. 1997 in Cellular and Molecular Immunology, ch 21, 450-459).
Tat is released from cells at relatively high levels and can be detected in the serum of HIV infected individuals. It is rapidly taken up by other cells through binding mediated by the basic domain of the molecule (Ma, M. and Nath, A. 1997 J. Virol. 71:2495). This feature allows extracellular Tat to transactivate latent HIV in other cells (Frankel, A. D. and Pabo C. O., 1998 Cell 55:1189; Li, C. J. et al. 1997 Proc. Natl. Acad. Sci. USA 94:8116). It also allows for Tat to transactivate other cellular processes, causing altered intracellular signaling in a variety of cell types (Li, C. J. et al. 1997 Proc. Natl. Acad. Sci. USA 94:8116; Viscidi, R. P. et al. 1989 Science 246:1606; Li, C. J. et al. 1995 Science 268:429; Westendorp, M. O. et al. 1995 Nature 375:497; Kolesnitchenko, V. et al. 1997 J. Virol. 71:9753). A consequence of these events is severe immunosuppressive. However, most of these studies have been conducted in vitro at nanomolar concentrations of Tat, and it is unclear to what extent this occurs in vivo. Extracellular Tat also causes rapid calcium fluxes in neurons and other cell types (Nath, A. et al. 1996 J. Virol. 70:1475; Nath, A. et al. 1996 J. Neurovirol. 2:17; Holden, C. P. et al. 1999 Neuroscience 91:1369; Haughney, N. J. et al. 1998 J. Neurovirol. 4:353; Nath, A. et al. 2000 Ann. Neurol. 47 (January 2000); Haughney, N. J. et al. 1999 J. Neurochem. 73:1363).
Interestingly, this rapid internalization of Tat also results in the efficient entry of Tat into the MHC class I processing pathway (Moy, P. et al. 1996 Mol. Biotechnol. 6:105; Fawell, S. et al. 1994 Proc. Natl. Acad. Sci. USA 91:664; Kim, D. T. et al. 1997 J. Immunol. 159:1666). In fact, a single peptide consisting of amino acids 49 to 57 of Tat was shown to facilitate the transport of ovalbumin (OVA) into the class I pathway (Moy, P. et al. 1996 Mol. Biotechnol. 6:105). Thus, the internalization of Tat and entry into the class I pathway would be expected to enhance the development of cytotoxic T-lymphocyte (CTL) responses aimed at Tat epitopes, if its transactivating activity were blocked. Inactivation of the transactivation responses aimed at Tat epitopes has been achieved by mutating residue Cys 22 to Gly. The mutated Tat functions as a dominant negative (Rossi, C. et al. 1997 Gene Ther. 4:1261; Caputo, A. et al. 1996 Gene. Ther. 3:235).
Several studies have shown that Tat is immunosuppressive. Tat can induce apoptosis in T cells by a process that involves activation of cellular cyclin dependent kinases (Li, C. J. et al. 1995 Science 268:429). Recently, Tat has been shown to up-regulate FasL on macrophages (Wu, M. X. and Schlossman, S. F. 1997 Pro. Natl. Acad. Sci. USA 94:13832; Dockrell, D. H. et al. 1998 J. Clin. Invest. 101:2394) and T cells (Westendorp, M. O. et al. 1995 Nature 375:497), resulting in apoptosis of the Fas expressing T cells. On the other hand, Tat has been shown to prevent apoptosis in some systems (Zauli, G. et al. 1993 Cancer Res. 53:4481). However, most of these studies have been conducted in vitro, and it is unclear to what extent Tat is immunosuppressive in vivo. A recent study by Cohen et al. (Cohen, S. S. et al. 1999 Proc. Natl. Acad. Sci. USA 96:10842) has shown that immunization using Tat in complete Freund's adjuvant (CFA) suppressed the response to a co-administered antigen, and that this effect was abrogated by oxidation of the Tat molecule. It would be desirable to evaluate the immunosuppressive properties of Tat in an in vivo system.
Tat from the human immunodeficiency virus type 1 (HIV-1) is an RNA binding transcriptional protein that is expressed early in HIV infection, and is necessary for high level expression of viral proteins (Garber, M. E. and Jones, K. A. 1999 Curr. Opin. Immunol. 11:460). Tat comprises 86 to 102 amino acids that are encoded by 2 exons, and Tat protein comprise five functional domains (Bayer, P. et al. 1995 J. Mol. Biol. 247:529). The first 72 amino acids are encoded by exon 1 and exhibit full transactivating activity. The amino terminal domain spans the first 21 amino acids; the cysteine-rich domain spans amino acids 22-37 and represents the transactivation domain; the basic domain spans amino acids 49-72, and contains the nuclear localization signal sequences, which facilitate the binding of Tat to Tat-responsive elements as well as the uptake of Tat by the cell (Jones, K. A. and Peterlin, B. M. 1994 Ann. Rev. Biochem. 63:717; Chang H. C. et al. 1997 Aids 11:1421; Barillari, G. et al. 1993 Proc. Natl. Acad. Sci. USA 90:7941). The second exon encodes the amino acid C-terminal sequence, which varies among different strains of HIV-1 from amino acids 73 to 86 or 73 to 102. The C terminus is not required for transactivation but does contain an RGD (arginine-glycine-aspartate) motif, which is important in binding to cell surface molecules and the extracellular matrix (Chang H. C. et al. 1997 AIDS 11:1421). The inventors have shown that the second exon of Tat influences the tertiary configuration of Tat and greatly potentiates Tat uptake (Ma, M. and Nath, A. 1997 J. Virol. 71:2495).
Tat is an unusual transcription factor as it can be released from cells and enter cells, while retaining its transactivating activity, which enables it to up-regulate a number of genes. It appears that the basic domain of Tat is important, not only for translocation and for nuclear localization but also for trans-activation of cellular genes. As such, targeting of Tat protein or, more simply, the basic protein provides great scope for therapeutic intervention in HIV-1 infection.
Modern vaccines typically consist of either a killed (inactivated) or a live, nonvirulent (attenuated) form of an infectious agent. Traditionally, the infectious agent is grown in culture, purified, and either inactivated or attenuated without losing the ability to evoke an immune response that is effective against the virulent form of the infectious organism. Notwithstanding the considerable success that has been achieved in creating effective vaccines against numerous diseases, AIDS is one disease that is not preventable through the use of traditional vaccines.
The task of developing an effective vaccine for immunoprophylaxis against HIV has been complicated by the genetic potential of the virus for great antigenic variability. This effort has largely been directed to proteins of the virus that are expressed on the surface of infected cells, which are recognized by cytotoxic T cells. The T cell response eliminates infected cells, while free virus is blocked and cleared by antibodies to surface antigens of the viron. Limitations of this mode of vaccination are readily apparent in HIV-1, which has demonstrated a great diversity in immunogenic viral epitopes and rapid mutational variations that occur within and between infected individuals.
On the other hand, intracellular Tat is efficiently processed by major histocompatibility complex (MHC) class 1 for presentation to cytotoxic T lymphocytes (CTL). CTL responses have been detected repeatedly in individuals infected with HIV (van Baalen, C. A. et al. 1997 J. Gen. Virol. 78:1913; Venet, A. et al. 1992 J. Immunol. 148:2899; Froebel, K. S. et al. 1994 AIDS Res. Hum. Retroviruses 10:S83; Ogg, G. S. et al. 1998 Science 279:2103), and other studies have shown that the presence of anti-Tat CTL during the initial phase of infection, correlates inversely with the progression of the infection to AIDS disease (Re, M. C et al. 1995 J. Aquir. Immune Defic. Syndr. Hum. Retrovirol. 10:408). Tat also shows very little variation between HIV subtypes, and the first exon is highly conserved among the different subtypes, except the O subtype (Gringeri, A. et al. 1998 J. Hum. Virol. 1:293). Because of these properties, Tat is an attractive candidate as a vaccine. Current studies on the development of a Tat vaccine utilize inactivated Tat “toxoid” in an effort to prevent the toxic and immunosuppressive effects of Tat (Gringeri, A. et al. 1998 J. Hum. Virol. 1:293; Gallo, R. C. 1999 Proc. Nat. Acad. Sci. USA 96:8324; Gringeri, A. et al. 1999 J. Aquir. Immune Defic. Syndr. Hum. Retrovirol. 20:371). Tat toxoid has been administered in incomplete Freund's adjuvant to HIV seronegative people, and has been shown to safely induce modest antibody and DTH responses (Gringeri, A. et al. 1998 J. Hum. Virol. 1:293; Gringeri, A. et al. 1999 J. Aquir. Immune Defic. Syndr. Hum. Retrovirol. 20:371). However, the use of a denatured molecule may destroy important epitopes and prevent Tat from efficiently entering cells for an optimum immune response. Also, current immunization strategies would not be expected to induce T helper 1 (Th1), or CTL responses, which are critical for antiviral immune responses. Biologically active Tat has been used to immunize monkeys (Cafaro, A. et al. 1999 Nat. Med. 5:643). This vaccination protocol achieved partial protection against the highly pathogenic SHIV virus. However, it is conceivable that had the immunosuppressive effect of Tat been abolished, a better immune response could have been attained.
The present inventors have discovered that Tat produced by recombinant methods tightly binds bacterial RNA, which conventional methods of purification of recombinant Tat are unable to remove from the protein. This tightly bound RNA tends to mask antigenic sites (epitopes) on the Tat protein. Stimulation of the immune system by recombinant Tat protein is thereby attenuated, which in turn reduces the Tat protein's usefulness as a vaccine.
The present inventors have also discovered that highly purified Tat does not cause immuno-suppression when given as a vaccine in mice, while still inducing a strong immune response. Moreover, recombinant Tat has heretofore been purified by reverse-phase HPLC, which gives rise to denaturation of the protein and concomitant loss of important epitopes. Thus, the conventional purification methodology results in less-than-optimal immunogenic recombinant Tat protein.
Heretofore, Tat has been produced by synthetic procedures. While the primary structure (amino acid sequence) of Tat can be attained by such methodology, the harsh chemical conditions required for such synthesis tend to interfere with protein folding. Thus, it has not been heretofore possible to faithfully produce a Tat protein by synthetic methods that possess naturally occurring Tat protein's tertiary structure. As a result, purely synthetic methods tend to produce Tat protein that lacks some or all of the epitopes that are present in naturally-occurring Tat protein, again resulting in less-than-optimal immune stimulation by recombinant Tat protein.
There is thus a need for a method for producing a recombinant Tat protein that possesses a tertiary structure that has not been compromised by harsh synthetic chemicals.
There is also a need for a method for producing recombinant Tat protein that is free of masking of antigenic sites by bacterial RNA.
There is also a need for a recombinant Tat protein that possesses the ability to be internalized by cells and that is not immunosuppressive.
There is also a need for a recombinant Tat protein that is processed via the MHC class I pathway.
There is also a need for an effective immunogen and a method for effectively eliminating Tat-expressing cells by evoking strong Th1 and CTL responses, and resulting in an effective vaccine against HIV.
Furthermore, protein-based vaccines, such as recombinant Tat, are often more effective if administered with at least one adjuvant to enhance their potency (T. W. Baba, V. Liska, A. H. khimani, N. B. Ray, P. J. Dailey, D. Penninck et al., Nat. Med. 1999 5 194-203). Unfortunately, after decades of research, insoluble aluminum salts. (generally called as “Alum”) still represent the only approved adjuvants for human use in the US (R. K. Gupta, G. R. Siber, 1995 13 1263-1276). Alum has been used as vaccine adjuvant for many years. However, it is not a potent adjuvant for recombinant proteins, and more importantly, it does not help in cell-mediated immune responses (R. K. Gupta, E. H. Relyveld, E. B. Lindblad, B. Bizzini, S. Ben-Efraim, C. K. Gupta, Vaccine. 1993 11 293-306). It is well known that, with protein-based vaccines, Alum as adjuvant only helps humoral immune responses, characterized by enhanced antibody production and the type-2 CD4 T helper cell (Th2) responses, such as enhanced release of cytokines like interleukin 4 (IL-4) and/or the enhanced production of IgG subtype IgG1 (R. K. Gupta, G. R. Rost, E. Relyveld, G. R. Siber, Adjuvant properties of aluminum and calcium compounds, In: M. F. Powell, M. J. Newman (Eds.), Vaccine design: the subunit and adjuvant approach, Plenum Press, New York, 1995, p 229-248). Therefore, there exists a clear need to develop alternative and improved vaccine adjuvants and/or delivery systems, especially those that can help in cell-mediated immune responses, for protein-based vaccines, such as recombinant Tat protein.
Over the last several decades, many other potential vaccine adjuvants have been developed (M. Singh, D. T. O'Hagan, Nat. Biotech. 1999 17 1075-1081). Some of them were proven to help in cell-mediated immune responses. Lipid A is one example. The adjuvant effect of the lipopolysaccharide (LPS) from Salmonella Minnesota R595 (Re) was first described as early as in 1956 (J. T. Ulrich, K. R. Myers. Monophosphoryl lipid A as an adjuvant: past experiences and new directions. In: Vaccine design: The subunit and adjuvant approach, Ed (M. F. Powell, M. J. Newman) Plenum Press, New York, N.Y. 1995 p 495-524). The lipid A region of the LPS was found to be responsible for the adjuvanticity. Lipid A, which generally aids in Th1-type responses, enhances immune responses primarily through its ability to activate antigen-presenting cells (APC) and to induce the release of cytokines such as interferon-gamma (IFN-γ) and IL-2. The strong toxicity of lipid A promoted the development of the detoxified MPL, which retains the adjuvant properties of lipid A but with much reduced side effects (A. J. Johnson, Adjuvant action of bacterial endotoxins on the primary antibody response. In: M. Landy, W. Braun, (Eds.) Bacterial endotoxins. New Brunswick: University Press, 1964 pp 252-262; J. R. Baldridge, R. T. Crane, Monophosphoryl lipid A (MPL) formulations for the next generation of vaccines, Methods 1999 19 103-107). Besides the immunostimulatory molecules such as the lipid A, particulates as vaccine adjuvants have been evaluated for many years (D. T. O'Hagan, M. Singh, R. K. Gupta, Adv. Drug. Del. Rev. 1998 32 225-246). Particulates, such as emulsions, microparticles, ISCOMs, liposomes, virosomes, and the virus-like particles (VLP), have comparable dimensions to the pathogens the immune system evolved to combat. Therefore, it is reasonable to use particulates as a vaccine delivery system. One of the most extensively investigated is the poly (lactide-co-glycolide) (PLGA) microparticle. It has proven to be a potential vaccine adjuvant and/or delivery system for years (D. T. O'Hagan, J. Pharm. Pharmcol. 1998 59 1-10). Usually, vaccines were incorporated into the microparticles for delivery (O'Hagan, 1998). However, vaccines can also be adsorbed on the microparticles (J. Kreuter, P. P. Speiser, Infect. Immun. 1976, 13: 204-210). For example, Kazzaz et al. (2000) recently demonstrated that PLGA microparticles with adsorbed HIV-1 p55 gag protein on their surface were capable of inducing potent cell-mediated immune responses, including CTL, in mice following intramuscular immunization (J. Kazzaz, J. Neidleman, M. Singh, G. Ott, D. O'Hagan, J. Control. Rel. 2000 67 347-356). Surface adsorption of vaccines on microparticles has advantage in that it avoids the damages to vaccines caused by the sonication and high-torque mechanical mixing often needed in the process of microparticle preparation. In addition, limitation caused by the slowness of vaccine release once being incorporated can also be avoided. Singh et al. (M. Singh, M. Briones, G. Ott, D. O'Hagan, Proc. Natl. Acad. Sci. 2000 97 811-816) reported that the size of PLGA microparticles with adsorbed pDNA directly related to the strength of the resulting immune response; wherein the relative ratio was 300 nm>1 micron>30 microns. The authors attributed this particle size relationship to the enhanced ability of the smaller particles to be taken up by antigen presenting cells. Nevertheless, particles less than 300 nm were not investigated by the authors most likely since 300 nm particles that could be produced using the process described by the authors.
As such, there is a need for a method to engineer nanoparticles less than 300 nm and even less than 100 nm using a rapid and reproducible one-step process that may be contained in one vessel wherein said nanoparticles can be used to more efficiently target protein antigen to antigen presenting cells. There is also a need for an effective adjuvant and/or delivery system for Tat to enhance both humoral and cellular Th1-type immune responses.